Fig. 6.
Cells from PP are less sensitive to ATP than MLN cells. Cells from PP and MLN were isolated and adjusted to 2 × 105 cells/sample, stained with Mac-1(CD11b) (a) or CD3 (b) antibodies, processed for permeabilization assay and incubated with crescent concentrations of ATP for 10 min at 37°C. During the last 5 min of incubation EB was added and samples analyzed by flow cytometry. Macrophages and lymphocytes were selected based on antibody staining. c Confocal microscopy of cytospin preparations showing relative distribution and levels of P2X7 receptor (red) and CD11b (green) in mononuclear cells freshly isolated from MLN, peritoneum and PP. Analysis of protein staining showed P2X7 receptor densities are greater in MLN than in peritoneal or PP cells and a number of cells do not colocalize with CD11b. This observation may explain why the MLN cells, particularly the lymphocytes, are more sensitive to ATP permeabilization. Nuclei stained with DAPI (blue). Representative of 3 samples from each site. Scale bars represent 20 µm. Data are representative of 3 experiments. * p < 0.05, ** p < 0.001, *** p < 0.0001.